Description of complex forms of a porin in Bacteroides fragilis and possible implication of this protein in antibiotic resistance

Periodic surveys of antibiotic susceptibility patterns among anaerobes have emphasized that new mechanisms of resistance have emerged, especially in t he Bacteroides fragilis group. Resistance to the combination of amoxicillin and clavulanic acid among some imipenem-susceptible Bacteroides fragilis s trains has been associated with modifications in outer membrane protein ele ctrophoretic patterns with the loss of some porin-like proteins. Porins. ar e outer membrane proteins that play a major part in membrane permeability; if they are under-expressed, they can be responsible for antibiotic resista nce. In a previous work, we isolated one outer membrane protein of 45 kDa f rom Bacteroides fragilis and showed its porin activity In the present study , we aim to isolate the different complex forms of this protein and to unde rline their possible role in antibiotic resistance. We therefore compared t he electrophoretic patterns of the outer membrane proteins of several strai ns of Bacteroides fragilis. Although these patterns are similar to each oth er, some proteins, especially those of high molecular weight, are less visi ble in the samples heated before electrophoresis. We targeted these high mo lecular weight proteins (which appeared sensitive to heat) and isolated the m by electro-elution. We thus identified two high molecular weight proteins (210 and 130/135 kDa) which seemed to be components of a complex including the 45 kDa outer membrane protein formerly identified by us as a porin pro tein. Their porin activities were tested by the swelling assay of proteolip osomes. which showed that the 210 kDa protein behaved like the 45 kDa prote in whereas the 130/135 kDa protein had less porin activity. Furthermore, sw elling assays with antibiotic solutions made it possible to compute the rol e of this protein complex in antibiotic resistance. (C) 2001 Academic Press .